Assessment Of Circulating Endothelial Cells And Their Progenitors As Potential Biomarkers Of Disease Activity And Damage Accrual In Behçet's Syndrome

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O R I G I N A L R E S E A R C H

Assessment Of Circulating Endothelial Cells And

Their Progenitors As Potential Biomarkers Of

Disease Activity And Damage Accrual In Behçet

’s

Syndrome

This article was published in the following Dove Press journal: Open Access Rheumatology: Research and Reviews

Alberto Floris Matteo Piga Silvia Pinna

Maria Maddalena Angioni Mattia Congia Piero Mascia Elisabetta Chessa Ignazio Cangemi Alessandro Mathieu Alberto Cauli

Rheumatology Unit, AOU University Clinic and University of Cagliari, Cagliari, Italy

Purpose: To explore the potential role of circulating endothelial cells (CECs) and their progenitors (EPCs) as biomarkers of disease activity and damage accrual in patients with

Behçet’s syndrome (BS), by using a standardised and reliable ﬂow cytometry protocol.

Patients and methods: CECs and EPCs were assessed in 32 BS patients and 11 gender/age/

smoking habits matched healthy controls (HC). They were identiﬁed by ﬂow cytometry as alive/

nucleated/CD45-negative/CD34-bright/CD146-positive and alive/nucleated/CD45-negative/ CD34-bright/CD309-positive events, respectively. In BS patients, demographic and clinical

features, including disease activity (assessed by Behçet’s disease current disease activity form,

BDCAF) and irreversible damage accrual (by the vasculitis damage index, VDI) were recorded. Uni- and multivariate analysis were performed to compare the CECs and EPCs concentrations in BS vs HC and to identify potential associations with demographic or clinical features.

Results: The CECs concentration was signiﬁcantly higher in the BS patients than HCs

[median (IQR) 15.0 (7.5–23.0) vs 6.0 (2.0–13.0) CECs/mL, p=0.024]. In BS patients, no

signiﬁcant associations were found between CECs and demographic features, present and

past clinical manifestations, BDCAF score and ongoing treatment. A signiﬁcant association

was observed between CECs and organ damage, as assessed by the VDI (rho 0.356, p=0.045). Higher levels of CECs were especially associated with vascular damage [median

(IQR) 23.0 (14.0–47.0) vs 13.0 (6.0–19.0) CECs/mL, p=0.011], including arterial aneurysm

and stenosis, complicated venous thrombosis, cerebrovascular accident. The concentration of

EPCs did not signiﬁcantly differ between the BS and HC [median 26.5 (13.0–46.0) vs 19.0

(4.0–42.0) EPCs/mL, p=0.316] and no signiﬁcant associations were observed between their

levels and any clinical characteristic.

Conclusion: Our study suggests that the CECs concentration is signiﬁcantly higher in BS

than healthy subjects, and it mainly correlates with vascular damage. A longitudinal exten-sion of the present study on a wider cohort would be useful to validate the potential role of CECs as a marker or, hopefully, predictor of vascular damage in BS.

Keywords: circulating endothelial cells, CECs, endothelial progenitor cells, EPCs, Behçet’s

syndrome

Introduction

Behçet’s syndrome (BS) is a multisystem relapsing inﬂammatory disorder, char-acterized by oral and genital aphtosis, skin lesions, uveitis, and potential vascular, neurologic and gastrointestinal involvement.1 The etiology of BS remains Correspondence: Matteo Piga

Rheumatology Unit, University Clinic AOU of Cagliari, SS 554, Monserrato, CA 09042, Italy

Tel +390706754069 Fax +39070513157 Email matteopiga@unica.it

Open Access Rheumatology: Research and Reviews

Dove

press

open access to scientiﬁc and medical research

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Open Access Rheumatology: Research and Reviews downloaded from https://www.dovepress.com/ by 93.71.243.115 on 23-Oct-2019

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unknown, although the most widely held pathogenic hypothesis is that of aberrant autoinﬂammatory response triggered by an environmental agent in genetically suscep-tible subjects.2,3Supporting this is the geoepidemiology of the disease and the association with polymorphisms in the HLA complex, particularly the HLA-B*51.4–6

Histopathologic studies demonstrated that the predomi-nant lesion in BS is vasculitis, affecting both the vessel wall and perivascular tissues. Leukocytoclastic vasculitis, fibrinoid necrosis of postcapillary venules, or perivascular neutrophilic accumulations are some of the reported find-ings in the early stages of the cutaneous lesions.7 Similarly, neutrophil infiltrate and endothelial cell activa-tion are recorded in the vasa vasorum of patients with BS and major vessels involvement.8

Circulating endothelial cells (CECs) and their progeni-tors (EPC) are restricted subpopulations of peripheral blood cells involved in the endothelial homeostasis.9 CECs are characterized by mature endothelial features and detach from vessel walls following vascular damage or the physiological tissue turnover.10EPCs, characterized by an immature phenotype, are bone marrow resident cells, mobilized upon speciﬁc stimulation. Once in the bloodstream, EPCs home to target tissues where they are involved in endothelial repair or remodeling.9

Abnormalities in CECs and EPCs concentrations have been recorded in several vasculitides.11 Woywodt et al found that the concentrations of CECs were higher in patients with ANCA-associated vasculitis (AAV), when compared with healthy controls (HC) or patients with infections and other non-ANCA associated glomerulone-phritis. Further, a significant association between CECs levels and disease activity was recorded in these patients (rho 0.704, p>0.0001).12 Similarly, in Kawasaki disease, the mean number of CECs was found to be significantly higher in patients with vasculitis than in HC, especially in the acute and subacute phases.13 In regard to EPCs, Závada et al found that patients with AAV have a sig-nificant and persistent deficiency of circulating EPCs when compared with HC, assuming an impaired mechan-ism of vascular repair that may contribute to repeated relapses in these patients.14 On the other hand, Nakatani et al found higher levels of EPCs following an increase in CECs in Kawasaki disease, particularly in patients with complicated coronary artery lesions, suggesting that EPCs may be involved in the repair of endothelial damage.13

Poor and contrasting data on abnormalities in CECs and EPCs, derived from inadequate methodologies, are currently available in BS.15,16

This study aimed to evaluate whether the concentrations of CECs and EPCs, as assessed by a standardized ﬂow cytometry protocol, are increased in patients affected by BS and signiﬁcantly correlate with clinical features. It would provide explorative data on the vascular involvement in the disease pathogenesis and the potential role of these cells as biomarkers of disease activity or damage accrual.

Methods

Patients And Controls

In this cross-sectional cohort study, 32 unselected consecutive adult patients diagnosed with BS according to the International Criteria for Behçet’s Disease (ICBD)17were recruited at the Rheumatology Unit of the University Clinic of Cagliari. Further, 11 gender, age, and smoking habits matched HC were investigated. Subjects with infective, neoplastic or not BS-related cardiovascular disease were excluded.

The study was approved by the Local Ethical Committee (N. 2018/6028) and written informed consent was obtained from all subjects. All procedures were in accordance with the Good Clinical Practice standards and Helsinki Declaration.

Clinical Assessment

Demographic and clinical data, as well as ongoing treat-ment, were recorded for each patient at recruitment. Present and past clinical manifestations were categorized according to the ICBD criteria (oral aphtosis, genital ulcers, pathergy positivity, ocular, vascular central nervous system involvement), including also arthritis and gastro-intestinal involvement. Erythrocyte sedimentation rate and C-reactive protein (CRP) values were collected as labora-tory parameters. Disease activity was measured by using the Behçet’s Disease Current Activity Form (BDCAF), both as continuous (total BDCAF score) and binominal variable (active: BDCAF ≥1, inactive BDACF=0).18 The Physician Global Assessment (PGA) of disease activity on a 0 to 10 visual analogic scale was also recorded. Finally, according to the OMERACT (Outcome Measures in Rheumatology) recommendations, irreversible organ damage was also assessed in our study. However, as no speciﬁc damage assessment tools are currently available for BS, the vasculitis damage index (VDI) was used as a surrogate, being the most employed in other systemic

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vasculitides and being already applied in other cohorts including BS.19–21 Thus damage, defined as irreversible scars of previous disease activity or its treatment lasted for at least 3 months, was recorded both as a continuous variable, reflecting the overall extension of damage accrual (total VDI score) and binary variable, reflecting the pre-sence or abpre-sence of any irreversible damage (VDI≥1 and VDI=0, respectively).19For the purpose of the study, items included in the cardiovascular and peripheral-vascular sec-tions of the VDI, including also cerebrovascular were classified as vascular damage.

Collection Of Blood Specimen

Peripheral blood was drawn from the antecubital vein with 21 G needles in EDTA vacutainer tubes. The ﬁrst har-vested 3 ml tube was used to determine sample leukocyte count, in order to assess dual-plaform counting. It was excluded from the direct analysis of CECs and EPCs, to avoid counting cells derived from vascular damage caused by venipuncture.

CECs And EPCs Count

CECs determinations were performed within 4 hrs from the sample collection by a standardizedflow cytometry proce-dure, recently described elsewhere.10Briefly, blood volume containing 20×106 leukocytes underwent erythrocyte-lysis with 40 mL of Pharm Lyse solution (BD Biosciences), fol-lowed by centrifugation and wash with 2 mL of Stain Buffer containing bovine serum albumin (BD Biosciences). Surface staining was accomplished by adding the resuspended pellet of each sample to the CEC Lyotube kit (Becton Dickinson, Custom), consisting in a panel tube (CD146PE, CD34PE-Cy7, CD309AlexaFluor647, CD45APC-H7, 7AAD) and a control tube (IsotypePE, CD34PE-Cy7, IsotypeAlexaFluor647, CD45APC-H7, 7AAD), and 1 µM Syto-16 (Thermo Fisher Scientific, Eisai, Medipost – US) was added as liquid drop-in.10Samples incubated in the dark for 30 mins at 4°C were then washed and re-suspended in 1.5 mL of FACSFlow (BD Biosciences). CECs were defined as alive, nucleated, CD45-negative, CD34-bright, CD146-posi-tive events (Figure 1); whereas, EPCs as alive nucleated, CD45-negative, CD34-bright and CD309-positive cells.9,10

Figure 1 Flow cytometry identification of circulating endothelial cells (CECs). (A) Lympho-monocytes were gated in a forward scatter versus side scatter (FSC-A/SSC-A) dot plot. (B) Dead cells were excluded (7-AAD/SSC-A dot plot) and (C) nucleated events (Syto16pos) were selected (Syto16/SSC-A dot plot). (D) Events characterized by lympho-monocytes features, alive, nucleated, expressing bright levels of CD34 and CD45neg were identified. Analysis for CD146 surface expression both on a CD146/ CD45 (E) and CD146/CD34 (F) dot plot was performed. The CEC compartment (CD34bright/CD45neg/CD146pos cells) was identified. Gates were drawn based on the signal of the control tube, containing the corresponding isotype control in combination with all the remaining surface reagents.

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Finally, 2.5×106events/sample with lympho-monocyte mor-phology were acquired by ﬂow cytometry (FACSCanto II, BD Biosciences). A threshold combination was set on FSC and FITC channel (Syto16) to exclude very small and non-nucleated events. Data were analyzed using FACSDiva v 8.0.1 (BD) software.

Statistical Analysis

Standard descriptive statistics were used to summarize the demographic and clinical features of the studied cohort. Continuous data were expressed as mean±SD and/or med-ian (IQR). t-Student test and Mann–Whitney U-test were used, when appropriate, to assess the association of CECs and EPCs with dichotomic clinical variables. Spearman’s rank correlation coefﬁcient was calculated to estimate the association between CECs and EPCs and continuous clin-ical variables. Linear multiple regression was used to further assess the independent association of baseline clin-ical features and levels of CECs and EPCs. P-values <0.05 were regarded as statistically signiﬁcant. All analyses were performed using SPSS version 24.

Results

Patients And Controls

Details on demographic and clinical data from the study cohort are reported inTable 1. As expected, no signiﬁcant differences were recorded between BS and HC groups, in terms of gender distribution (males 53.1% and 45.5%, respectively, p=0.929), age at recruitment (mean 49.6 ±11.3 and 44.8 ±12.6, p=0.239), and smoking habits (19.6% and 18.2%, p=1.000).

CECs

The number of CECs was signiﬁcantly higher in BS patients [median (IQR) 15.0 (7.5–23.0), range 0–48 CECs/mL] than HC [median (IQR) 6.0 (2.0–13.0), range 0–18] CECs/mL (p=0.024) (Figure 2). The association of CECs levels with BS was conﬁrmed after correction for sex, age, and smoking habits (p=0.040).

Within the BS group, no signiﬁcant associations were found between CECs and demographic features, past or present clinical manifestations, BDCAF, PGA, and ongoing treatment (p>0.05) (Table 2). CECs levels signif-icantly correlated with the VDI, both in terms of total score (Spearman’s coefﬁcient 0.356, p=0.045) and occur-rence of any damage [median (IQR) 19.0 (13.0–27.0) vs

Table 1 Baseline Features

BS (n=32) HC (n=11) p

Demographics

Males 17 (53.1%) 5 (45.5%) 0.929

Mediterranean Europe origin 30 (93.8%) 11 (100%) 1.00

Assessment age 49.6 (11.3) 44.8 (12.6) 0.239 Onset age 33.5 (10.5) – Diagnosis age 37.5 (11.3) – Disease duration 12.2 (10.6) – HLA-B51 7/19 (36.8%) -Current smoker 6 (19.4%) 2 (18.2%) 1.00

Ever occurred manifestations

Oral aphtosis 32 (100%) -Genital aphtosis 22 (68.8%) -Skin lesions 26 (81.3%) -Ocular involvement 17 (53.1%) -CNS lesions 3 (9.4%) -Vascular lesions 7 (21.9%) -Pathergy test 4 (12.9%) -Arthritis 11 (34.4%) -GI involvement 1 (3.1%) -Active manifestations Oral aphtosis 4 (12.5%) Genital aphtosis 0 (0%) Skin lesions 5 (15.6%) Ocular involvement 2 (6.3%) CNS lesions 0 (0%) Vascular lesions 1 (3.1%) Pathergy test 0 (0%) Arthritis 0 (0%) GI involvement 1 (3.1%) ESR 19.0 (19.5) CRP 4.0 (4.5) Clinimetric indices BDCAF score 2.3 (2.8) -BDCAF≥1 16 (50.0%) -VDI score 1.0 (1.6) -VDI≥1 17 (53.1%) -PGA 2.75 (2.41) -Treatment Colchicine 8 (25%) -Glucocorticoids 22 (68.8%) -Conventional immunosuppressant 15 (46.8%) Azathioprine 13 (40.6%) Methotrexate 1 (3.1%) Thalidomide 1 (3.1%) Biologic immunosuppressant 6 (18.6%) -TNF inhibitor 5 (15.6%) IL1R inhibitor 1 (3.1%)

Notes: Categorical variables are expressed as number (%); continuous variables as mean (SD).

Abbreviations: BS, Behçet’s syndrome; HC, healthy controls; CNS, central ner-vous system; GI, gastrointestinal; BDCAF, Behçet’s Disease Current Activity Form; VDI, vasculitis damage index; PGA, Physician Global Assessment; TNF, tumour necrosis factor; IL1R, interleukin 1 receptor.

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12.0 (6.0–16.0) CECs/mL in VDI ≥1 and VDI=0, respec-tively; p=0.027] (Figure 3).

Higher levels of CECs were especially associated with vascular damage [median (IQR) 23.0 (14.0–47.0) vs 13.0 (6.0–19.0) CECs/mL, p=0.011], including arterial aneur-ysm, arterial stenosis, complicated venous thrombosis, cerebrovascular accident.

EPCs

The concentration of EPCs did not signiﬁcantly differ between the BS [median (IQR) 26.5 (13.0–46.0), range 0–310 EPCs/mL] and HC groups [median (IQR) 19.0 (4.0–42.0), range 0–162 EPCs/mL, p=0.316] (Figure 4).

Within the BS group, the highest value of EPCs (310 EPCs/mL) was recorded in a patient who had ongoing deep venous thrombosis on one lag. On univariate analy-sis, EPCs concentrations signiﬁcantly correlated with recruitment age (rho 0.361; p=0.042), disease duration (0.377, P=0.036), and CRP (0.408; p=0.03) (Table 2). However, these associations were not conﬁrmed in multi-variate analysis.

Discussion

BS is a complex multisystem disease, where the wide heterogeneity in clinical presentation and the lack of reli-able biomarkers often result in signiﬁcant difﬁculties in terms of diagnosis, monitoring and prognostic assessment. On the basis of the experience gained in other vasculitides, this study aimed to provide reliable data on the potential role of CECs and EPCs as biomarkers of disease activity or irreversible damage accrual in BS.

In our cohort, we found a significantly increased num-ber of CECs in BS patients when compared with HC. However, no significant associations with current disease activity or other specific clinical patterns were recorded, with the exception for vascular irreversible damage. These findings suggest that, in the subset of patients with vascu-lar involvement, the endothelial dysfunction with detach-ment of CECs persists even during the inactive periods. On the other hand, the lack of significant association with disease activity may be due to that all recruited patients were already on treatment, a few of them were on high disease activity and only a subject had a vascular active manifestation.

In a previous study, Kulty et al found that BS patients, especially in the active period, have elevated numbers of CECs compared with healthy controls. Conversely, in other studies, where BS was included among other vasculitides, no increase in CECs concentration was demonstrated.16

Compared with previous studies, ourfindings on CECs in BS are the first derived by a validated flow cytometry protocol. Indeed, most data on CECs are so far obtained by immunomagnetic separation protocols, where endothelial cells are isolated from whole blood with magnetic particles coated with anti-endothelial antibodies (eg, anti-CD146) and counted byfluorescence microscopy.11However, this methodology is affected by significant limits in terms of reliability caused by the inter-operator variability and the potential aspecific binding of leukocytes to beads.11In this context, a significant advance for the reliable identification of CECs was recently made with the validatedflow cyto-metry protocol proposed by Lanuti et al in 201810 and applied in our study, where CECs are defined as alive, nucleated, CD45-negative, CD34-bright, CD146-positive events.

In regard to the EPCs, in our BS cohort they were not significantly increased, and no significant associations were observed between their levels and any clinical char-acteristic. Further, no significant association was recorded between EPCs and CECs concentration. Similarly, Bozkirli et al22 observed that EPCs levels were compar-able in patients with BS and HC (p=0.849) and they did not correlate with disease activity or other clinical fea-tures, with the exception of thrombosis. Thus, our and previous data suggest that no abnormalities in repair sys-tem of endothelial damage are present in BS.

However, also for EPCs some methodological consid-erations are needed. Indeed, signiﬁcant issues exist on the best cell surface markers to distinguish EPCs from mature

Figure 2 Concentration of circulating endothelial cells (CECs) in Behçet’s syn-drome (BS) patients and healthy controls (HC). The ends of the box are the 1st and 3rd interquartile, the vertical line inside the box is the median, the whiskers represent the highest and lowest values.

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T able 2 Assessment Of Association Of CECs And EPCs Concentrations With Demographic And Clinical Features CECs p EPCs p Median (IQR)/rho coef ﬁ cient Median (IQR)/rho coef ﬁ cient Demo graphics Males 16.0 (8.0 –25.0) vs 13.0 (6.0 –18.0) 0.289 31.0 (15.0 –88.0) vs 26.0 (10.0 –42.0) 0.290 Assessment age 0.211 0.247 0.361 0.042* Onset age 0.053 0.781 0.095 0.618 Diagnosis age 0.035 0.854 − 0.004 0.985 Disease duration 0.200 0.281 0.377 0.036* HLA-B51 14.0 (6.0 –25.0) vs 13.5 (7.5 –24.5) 0.680 31.0 (12.0 –110.0) vs 27.0 (13.0 –67.0) 0.612 Curr ent smok er 9.0 (20 –19.0) vs 16.0 (8.0 –23.0) 0.227 23.5 (16.0 –46.0) vs 28.0 (10.0 –46.0) 0.783 Pre sent and past manifestations Oral aphtosis 15.0 (8.0 –20.0) vs -26.5 (13.0 –46.0) vs -Genital aphtosis 16.0 (8.0 –20.0) vs 13.5 (7.0 –20.0) 0.587 23.0 (12.0 –45.0) vs 29.0 (25.0 –109.0) 0.272 Skin lesions 15.0 (8.0 –25.0) vs 12.0 (6.0 –19.0) 0.302 26.5 (12.0 –59.0) vs 28.5 (20.0 –45.0) 0.904 Ocular inv olv ement 14.0 (6.0 –23.0) vs 16.0 (8.0 –20.0) 0.965 31.0 (20.046.0) vs 18.0 (3.0 –44.0) 0.136 CNS lesions 12.0 (6.0 –29.0) vs 16.0 (8.0 –23.0) 0.899 27.0 (14.0 –31.0) vs 26.0 (12.0 –46.0) 0.796 V ascular lesions 19.0 (13.0 –23.0) vs 14.0 (6.0 –20.0) 0.240 25.0 (10.0 –59.0) vs 27.0 –14.0 –46.0) 0.837 Pathergy test 15.5 (6.0 –24.5) vs 14.0 (6.0 –20.0) 0.824 11.0 (6.5 –62.0) vs 27.0 (14.0 –46.0) 0.409 Ar thritis 14.0 (8.0 –23.0) vs 16.0 (7.0 –23.0) 0.463 27.0 (6.0 –109.0) vs 26.0 (15.0 –45.0) 1.000 GI in volv ement 2.0 (2.0 –2.0) vs 16.0 (7.0 –23.0) 0.212 16.0 (16.0 –16.0) vs 27.0 (12.0 –46.0) 0.551 Active manifestations Oral aphtosis 22.0 (10.5 –26.0) vs 14.0 (7.5 –21.5) 0.754 14.0 (6.5 –30.0) vs 27.5 (14.5 –52.5) 0.190 Genital aphtosis -15.0 (7.5 –23.0) 0.475 -vs 26.5 (13.0 –46.0) -Skin lesions 13.0 (4.0 –19.0) vs 16.0 (8.0 –23.0) 0.475 15.0 (10.0 –16.0) vs 28.0 (14.0 –59.0) 0.113 (Continued )

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T able 2 (Continued). CECs p EPCs p Median (IQR)/rho coef fi cient Median (IQR)/rho coef fi cient Ocular inv olv ement 15.0 (13.0 –17.0) vs 15.0 (7.0 –23.0) 0.855 49.0 (10.0 –88.0) vs 26.5 (14.0 –46.0) 0.907 CNS lesions -vs 15.0 (7.5 –23.0) -vs 26.5 (13.0 –46.0) -V ascular lesions 14.0 (14.0 –14.0) vs 16.0 (7.0 –23.0) 0.832 310.0 (310 –310) vs 26.0 (12.0 –46.0) 0.093 Pathergy test -vs 15.0 (7.5 –23.0) -vs 26.5 (13.0 –46.0) -Ar thritis -vs 15.0 (7.5 –23.0) -vs 26.5 (13.0 –46.0) -GI in volv ement -vs 15.0 (7.5 –23.0) -vs 26.5 (13.0 –46.0) -ESR − 0.147 0.455 0.161 0.413 CRP 0.183 0.352 0.408 0.031* Clinimetric indices BDC AF score − 0.140 0.445 − 0.202 0.267 BDC AF ≥ 1 14.0 (7.0 –20.0) vs 16.0 (7.5 –26.0) 0.341 23.5 (11.0 –45.5) vs 26.5 (19.0 –52.5) 0.665 VDI score 0.356 0.045 0.252 0.163 VDI ≥ 1 19.0 (13.0 –27.0) vs 12.0 (6.0 –16.0) 0.027 31.0 (20.0 –59.0) vs. 16.0 (7.0 –42.0) 0.157 PGA 0.90 0.626 − 0.173 0.1343 T reatment Colchicine 12.0 (5.0 –19.0) vs 15.0 (8.5 –24.0) 0.241 43.0 (16.5 –77.5) vs 25.5 (13.0 –45.5) 0.408 Glucocortic oids 16.0 (6.0 –23.0) vs 13.5 (8.0 –20.0) 0.848 26.0 (12.0 –59.0) vs 27.5 (14.0 –44.0) 0.807 Con ve ntional immunosuppressant 13.5 (8.0 –23.0) vs 16.5 (6.0 –23.0) 0.976 18.0 (10.0 –26.0) vs 43.0 (18.0 –59.0) 0.678 Biologic immunosuppressant 23.0 (12.0 –29.0) vs 14.0 (7.0 –20.0) 0.340 27.5 (14.0 –31.0) vs 26.0 (12.0 –59.0) 0.329 Notes: Association of CECs and EPCs concentration with categorical variables was expr essed as mean (SD) concentration in patients with vs without the studi ed characteristic; association analysis was performed by t-Student test or Mann –Whitne y test. Association betw een concentration of CECs and continuous variables was expr essed with the rho Spearman coef ficient and respective signi ficance le vel. *Statistical signi ficance not con firmed in multivariate analysis. Abbre viations: BS, Behçet ’s syndr ome; CNS, central ner vous system; GI, gastr ointestinal; BDC AF , Behçet ’s Disease curr ent activity form; VDI, vasculitis damage index; PGA, Ph ysician Global Assessment.

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endothelial cells. EPCs are currently more commonly identiﬁed by ﬂow cytometry as CD34-positive, VEGFR2 (or CD309) positive, and CD133-positive cells,23 though many studies, including the present, typically employ two of the three receptors, mainly due to the controversial utility of CD133.24–26

The main strength of the present study is that it is the first work specifically focused on BS which assessed both CECs and EPCs concentration by using a standardized flow cytometry protocol, providing highly reliable data especially for CECs.

On the other hand, the main limits of the study were the small size of the cohort, especially in the control group, and the relatively low number of patients with

high disease activity. These factors might have prevented to detect significant differences in concentration of EPCs in BS patient and HC, as well as significant associations of CECs and EPCs with disease activity. Further, at present, the lack of a longitudinal assessment did not allow to evaluate the potential correlation between modifications in the concentration of these cells and treatment or changes in disease activity. However, in the context of a complete lack of valid biomarkers for BS, these explora-tive data provide a relevant contribute in understanding the potential role of these cells in BS and in planning future research in thefield.

In conclusion, our study suggests that the CECs con-centration is signiﬁcantly higher in BS than healthy sub-jects, and it correlates with vascular damage, even not currently active. A longitudinal extension of the present study on a wider cohort would be useful to validate the potential role of CECs as a marker or, hopefully, predictor of vascular damage in BS.

Abbreviations

CECs, circulating endothelial cells; EPCs, endothelial pro-genitor cells; BS, Behçet syndrome; HC, healthy controls; AAV, anti-neutrophil cytoplasmic antibody-associated vas-culitis; ICBD, International Criteria for Behçet’s Disease; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; BDCAF, Behçet’s Disease Current Activity Form; PGA, Physician Global Assessment; OMERACT, Outcome Measures in Rheumatology; VDI, vasculitis damage index.

Ethics Statement

The study was approved by the Local Ethical Committee of the Azienda Ospedaliero Universitaria di Cagliari (PG/ 2018/6028), and written informed consent was obtained from all subjects. All procedures were in accordance with the Good Clinical Practice standards and Helsinki Declaration.

Acknowledgments

The authors thank all recruited patients and control sub-jects for their participation in this study. We thank the Associazione Italiana Malattia Sindrome di Behçet (SIMBA) for partially funding this study and the Italian Society of Rheumatology (Società Italiana di Reumatologia - SIR) for supporting by a bursary to the author Alberto Floris.

Figure 3 Concentration of circulating endothelial cells (CECs) in patients affected by Behçet’s syndrome with at least one item of irreversible damage and without any damage, as assessed by the vasculitis damage index (VDI). The ends of the box are the 1st and 3rd interquartile, the vertical line inside the box is the median, the whiskers represent the highest and lowest values.

Figure 4 Concentration of endothelial progenitor cells (CECs) in Behçet’s syn-drome (BS) patients and healthy controls (HC). The ends of the box are the 1st and 3rd interquartile, the vertical line inside the box is the median, the whiskers represent the highest and lowest values.

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Disclosure

The authors report no conﬂicts of interest in this work.

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